CN102346146A

CN102346146A - Quantification method of bacteriochlorophyll a containing microbes in sea

Info

Publication number: CN102346146A
Application number: CN2011102005612A
Authority: CN
Inventors: 焦念志; 张瑶; 赵子豪
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2011-07-18
Filing date: 2011-07-18
Publication date: 2012-02-08

Abstract

The invention relates to a microbe quantification method, and discloses a quantification method of bacteriochlorophyll a containing microbes (BCM) in sea. The method comprises steps that: a sample is pre-filtered, fixed, processed from DAPI staining, filtered, and prepared into a sample slice; the sample slice is observed by using an automatic epifluorescence microscope with a camera and a mercury lamp, different fluorescence excitation blocks are used for carrying out observation and shooting upon IR fluorescence, cyanobacteria red fluorescence and DAPI blue fluorescence images in a same visual field, all images are obtained through automatic exposure, and IR, Cyano and DAPI images are obtained on time series after the microscope is focused; cells in the images are identified and analyzed by using an Image-Pro Plus software, and novel two-dimensional digital images are obtained; in a same visual field, an initial time IR image, a plateau-phase Cyano image and an initial time DAPI image in the time series are respectively obtained, and corresponding two-dimensional digital images are obtained, such that the BCM abundance in the sea is obtained.

Description

The quantivative approach that contains the microorganism of bacteriochlorophyll in the ocean

Technical field

The present invention relates to the quantivative approach of a kind of microorganism; Especially the accurate quantivative approach that relates to the micropopulation that contains bacteriochlorophyll (Bchl.a) unique in a kind of marine environment of the formula fluorescence microscopy that falls based on time series observation, automated imaging and digital assay

Background technology

Microorganism (the BCM that contains bacteriochlorophyll; Bacteriochlorophyll a Containing Microbes) is one type of component in the marine environment microflora with specific function; Be used to make up its dynamics data owing to lack effective abundance counting, its ecological functions also are not very clear.Aerobic non-oxygen-production photoheterotrophy bacterium (AAPB, Aerobic Anoxygenic Phototrophic Bacteria) is one type of main monoid of BCM of understanding recently, has had report to show, circulation has important role to this type flora to marine carbon.The extensive distribution of complete understanding AAPB and unique function will be to being based upon the ocean energy that produces on the oxygen photosynthesis basis and the understanding generation far-reaching influence of oxygen balance at present.Yet all the time, because unicellular bacteriochlorophyll content is very low, and is subjected to chlorophyllous infrared interference, the accurate quantitative comparison difficulty of BCM.

So far; Method of counting with BCM in the Yu Haiyang mainly contains following several: the instantaneous dynamics technology of infrared fast-pulse speed (IRFRR) induced fluorescence (Kolber et al.; 2000), infrared formula fluorescence microscopy (IREM) (the Kolber et al. that falls; 2001), high performance liquid chromatography (HPLC) (Goericke; 2002), dual regulation technology (dual modulation techniques) (Kobl í zek et al.; 2005) and quantitative polyase chain reaction (QPCR) (Schwalbach and Fuhrman, 2005) etc.

In the above-mentioned the whole bag of tricks; No matter be the IRFRR technology of electron transport in the monitoring bacterial photosynthesis process; Still directly measure bacteriochlorophyll a and chlorophyll a content and ratio (BChl.a/Chl.a) the HPLC technology and based on the quantitative QPCR technology of photosynthetic gene the abundance of BCM in the ocean can not be provided directly all, realization is to microbial function crowd's direct measurement.With the Infrared fluorescence that BChl.a was sent out is that detection signal IREM technology then is to detect the easiest method (Kolber et al., 2001 of BCM abundance in the ocean; Schwalbach and Fuhrman, 2005).But practice finds, all contain chlorophyllous authotroph and all can be counted among the BCM by the IREM method, because they can both send Infrared fluorescence to a certain extent under the exciting of the suitable light of wavelength.And in the field sea water sample, the abundance of cyanobacteria (common genus is prochlorococcus and Synechococcus in the marine environment) has caused remarkable positive error (the Zhang and Jiao 2004 of IREM method even as big as disturbing the BCM counting; Schwalbach and Fuhrman, 2005).The applicant finds that in the check and analysis in the East Sea positive error of being introduced by Synechococcus in the shallow water territory can be up to 30% (Zhang and Jiao2007).Even if the red fluorescence image of using this type of cyanobacteria (Synechococcus) subtracts from the cell particle diameter or through difference infrared, the red fluorescence signal and carries out error correction, and by another kind of cyanobacteria---the error that prochlorococcus is introduced then can't be calculated.Because prochlorococcus not only particle diameter is similar with bacterial cell; And the Synechococcus (Synechococcus) of the red fluorescence signal that the is sent tool hyperfluorescence that can be coexisted in the seawater appearance covers, thereby under the initial cyanobacteria red fluorescence visual field, can't be observed.In fall the formula fluorescent microscope (EM) or the IREM method of classics; Observe fast in order to prevent Synechococcus fluorescent quenching; Cause when adopting initial cyanobacteria image to carry out error correction, and can't realize correction (the Zhang and Jiao 2004 of the gross error that prochlorococcus causes the BCM counting; Schwalbach and Fuhrman, 2005).

Summary of the invention

The objective of the invention is to provides a kind of new quantivative approach that can carry out containing in the accurate quantitative ocean microorganism of bacteriochlorophyll to BCM in the ocean to the coexist error of BCM counting in the caused ocean of the indeterminable Synechococcus of IREM method and prochlorococcus.

Technical scheme of the present invention is based on infrared formula fluorescence microscopy (the Time-series observation based cyanobacteria-calibrated Infrared Epifluorescence Microscopy of falling of the deduction cyanobacteria error of time series observation; TIREM); This method can accurately calculate the sum of Synechococcus and prochlorococcus, thereby through logical operation BCM in the ocean is carried out accurate counting again.

The present invention includes following steps:

1) with sample pre-filtering, fixing, DAPI (4 ' 6-diamidino-2-phenylindole, a kind of double-stranded DNA fluorescent dye) dyeing, filter and film-making;

2) with being furnished with beam coupling assembly (CCD) camera of infrared-sensitive and the formula fluorescent microscope that automatically falls of mercury lamp is observed; Respectively Infrared fluorescence (IR), cyanobacteria red fluorescence (Cyano) and the DAPI blue-fluorescence image of the same visual field are observed and taken pictures with different fluorescence excitation pieces; All images all obtain through automatic exposure; After the micro-focusing, carry out obtaining on IR, Cyano and the DAPI temporal sequence of images;

In step 2) in, said mercury lamp can adopt 100W mercury lamp (OLYMPUS U-LH100HGAPO); Saidly can adopt 3 groups of different fluorescence excitation pieces with different fluorescence excitation pieces; After the said micro-focusing, carry out obtaining on IR, Cyano and the DAPI temporal sequence of images and can whenever obtain an image, continue 10～15min at a distance from 60s.

3) (Media Cybernetics Inc.) discerns and analyzes the cell in the image, and obtains new binary digit image with Image-Pro Plus (IPP) software;

4) calculate: under an identical visual field; Obtain respectively initial time under the time series the IR image (be designated as image a), the Cyano image (being designated as image b) of plateau and the DAPI image (being designated as image c) of initial time; And obtain corresponding binary digit image (being designated as image d, image e and image f) respectively; The abundance of BCM in the ocean=Boolean AND[(d) then; (f)]-Boolean AND[(d); (e)], wherein " Boolean AND " refers to " logical operation with ".

In step 4), the Cyano image of said plateau is the maximum image of cyanobacteria number that obtains during time series is observed, and comprises Synechococcus and prochlorococcus simultaneously.

Advantage of the present invention is: the popularization of technology (formula that falls fluorescent microscope all provides in common laboratory); Operate easily and spend little; Can be on unicellular level with the ocean in other bacterium difference in BCM, Synechococcus and prochlorococcus and the sample come, thereby can carry out accurate counting to the BCM in the ocean.

Embodiment

1) with the institute sample thief earlier with the bolting silk pre-filtering of 20 μ m to remove big organic-biological body and particles of inorganic material; It is fixedly 15min of 2% paraformaldehyde (PFA) that final concentration is used in the back; Be the DAPI dyeing 30min of 5 μ g/ml with final concentration again, cell is filtered on black polycarbonate (PC) film (Whatman) in 0.2 μ m aperture afterwards.Clip 1/4 film appearance, cell faces up and places on the slide, drips to go up anti-quencher mirror oil, covered.

2) with the formula fluorescent microscope that automatically falls (OLYMPUS BX61) film-making is observed, used fluorescence excitation piece is as follows.The used fluorescence excitation piece (Chroma Technology Corp) of cell that contains Infrared fluorescence is: 350-550nm excites, LP 850nm emission, 650nm beam split (OLYMPUS U-MWIY2) (Kolber et al., 2001; Zhang and Jiao, 2004) (IR-settings).The cell that dyes DAPI is used for contrast, and its used fluorescence excitation piece is: 330-385nm excites, 420nm emission, 400nm beam split (OLYMPUS U-MWU2) (Kolber et al., 2001; Zhang and Jiao, 2004) (DAPI-settings).The used fluorescence excitation piece of cyanobacteria (mainly comprising Synechococcus and prochlorococcus) cell red fluorescence signal is: 530-550nm excites, and 475IFnm launches (Li and Wood, 1988; Sherry and Wood, 2001), 570nm beam split (OLYMPUS U-MWIG3) is (Cyano-settings).Each field of microscope oily sem observation of * 100 obtains infrared image (IR), cyanobacteria image (Cyano) and DAPI image simultaneously to the same visual field.All images all obtain through automatic exposure, and its " gain limit " (gain-limitation, in the automated imaging software is provided with parameter) is 8.Seasonal effect in time series IR, Cyano and DAPI image after initial about 30 seconds focus steps, the 10-15min that is hunted down continuously, the time interval is 60 seconds.

3) (Media Cybernetics Inc.) discerns and analyzes the cell in the image with Image-Pro Plus (IPP) software.At first regenerate gray level image, make the original image standardization through measuring the average background gray-scale value.Cell boundaries in the image is with " Variance filter " (difference filtration of IPP; One in the IPP software is provided with parameter) measure and strengthen, " Variance filter " can replace 3 * 3 neighbor pixel gray-scale values on every side with the standard deviation that gray-scale value calculates.The image that generates weakens with " 3 * 3neighborhood median filter " (3 * 3 adjacent median filter, in the IPP software is provided with parameter).Last software is with the automatic recognizing cells of a gray-scale value that fixedly installs, thus the acquisition binary picture.Point in the ccd image is carried out binary digitization to produce a new image, can obtain more credible comparable data.Background gray levels is " 0 " in this binary picture, and the point that each is identified is no matter size all is " 1 ".At last, the celluar localization between the different images can through logical operation " with " (AND) carry out.

4) calculate: under an identical visual field; Obtain the IR image a of initial time under the time series, the cyanobacteria image b of plateau (the maximum image of cyanobacteria number that time series obtains in observing respectively; Comprise Synechococcus and prochlorococcus simultaneously) and the DAPI image c of initial time; And obtain corresponding binary digit fractional analysis result images d, e and f respectively; Abundance=Boolean the AND[(d) that then contains the microorganism of bacteriochlorophyll in the ocean; (f)]-Boolean AND[(d), (e)].

Use the present invention in South China Sea, the East Sea; And the torrid zone, temperate zone, subfrigid zone have been carried out striding in the Pacific Ocean, the Atlantic and the Indian Ocean; The BCM check and analysis on a plurality of gradients along offshore to ocean, and to wherein main monoid---abundance of the photosynthetic different oxygen bacterium of aerobic non-oxygen-production (AAPB) and the ratio of shared total bacterium have been carried out ecological investigation.The result shows, it is about 30%～500% that this method can be corrected coastal waters 10%～60%, continental shelf, and ocean are up to 200%～10000% error of the first kind; Accurate abundance of the AAPB that is obtained and ratio data show that it distributes and the nutrition condition in sea area of living in is proportionate; Its ratio at offshore can be up to 16%; Then be low to moderate 1% even lower in zone, open ocean, before having revised about AAPB in ocean proportion up to 11% report.

Claims

1. contain the quantivative approach of the microorganism of bacteriochlorophyll in the ocean, it is characterized in that may further comprise the steps:

1) with sample pre-filtering, fixing, DAPI dyeing, filter and film-making;

2) with being furnished with the beam coupling assembly camera of infrared-sensitive and the formula fluorescent microscope that automatically falls of mercury lamp is observed; Respectively Infrared fluorescence, cyanobacteria red fluorescence and the DAPI blue-fluorescence image of the same visual field are observed and taken pictures with different fluorescence excitation pieces; All images all obtain through automatic exposure; After the micro-focusing, carry out obtaining on IR, Cyano and the DAPI temporal sequence of images;

3) with Image-Pro Plus software the cell in the image is discerned and analyzed, and obtain new binary digit image;

4) calculate: under an identical visual field; Obtain the DAPI image of IR image, the Cyano image of plateau and the initial time of initial time under the time series respectively; And obtain corresponding binary digit image respectively; The abundance of BCM in the ocean=Boolean AND[(d) then; (f)]-Boolean AND[(d); (e)], wherein " Boolean AND " refers to " logical operation with ".

2. contain the quantivative approach of the microorganism of bacteriochlorophyll in the ocean as claimed in claim 1, it is characterized in that in step 2) in, said mercury lamp adopts the 100W mercury lamp.

3. contain the quantivative approach of the microorganism of bacteriochlorophyll in the ocean as claimed in claim 1, it is characterized in that in step 2) in, saidly adopt 3 groups of different fluorescence excitation pieces with different fluorescence excitation pieces.

4. the quantivative approach that contains the microorganism of bacteriochlorophyll in the ocean as claimed in claim 1; It is characterized in that in step 2) in; After the said micro-focusing, carrying out obtaining on IR, Cyano and the DAPI temporal sequence of images is that every separated 60s obtains an image, continues 10～15min.

5. the quantivative approach that contains the microorganism of bacteriochlorophyll in the ocean as claimed in claim 1; It is characterized in that in step 4); The Cyano image of said plateau is the maximum image of cyanobacteria number that obtains during time series is observed, and comprises Synechococcus and prochlorococcus simultaneously.